Journal of the Arkansas Academy of Science Journal of the Arkansas Academy of Science

Volume 72 Article 21

2018

Prevalence of Cytauxzoon felis (Protista: Apicomplexa) in Feral Prevalence of Cytauxzoon felis (Protista: Apicomplexa) in Feral

Cats in Russellville Arkansas Cats in Russellville Arkansas

Cynthia H. Jacobs Arkansas Tech University, cjacobs@atu.edu

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Recommended Citation Recommended Citation Jacobs, Cynthia H. (2018) "Prevalence of Cytauxzoon felis (Protista: Apicomplexa) in Feral Cats in Russellville Arkansas," Journal of the Arkansas Academy of Science: Vol. 72 , Article 21. DOI: https://doi.org/10.54119/jaas.2018.7215 Available at: https://scholarworks.uark.edu/jaas/vol72/iss1/21

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Journal of the Arkansas Academy of Science, Vol. 72, 2018123

Prevalence of Cytauxzoon felis (Protista: Apicomplexa) in Feral Cats inRussellville Arkansas

C.H. Jacobs

Department of Biological Sciences, Arkansas Tech University, Russellville, AR 72801

Correspondence: cjacobs@atu.edu

Running title: Prevalence of Cytauxzoon felis in Feral Cats

Abstract

Cytauxzoon felis (C. felis) is a protozoanhemoparasite of domestic and wild felids. Transmittedby ixodid ticks, the sylvatic reservoir for this organismin North America is the bobcat (Lynx rufus) in which theinfection is apparently self-limiting. In domestic cats(Felis catus), C. felis causes a highly fatal disease witha distribution that covers much of the central,southcentral and southeastern U.S. and parallels that ofthe primary vector, the lone star tick (Amblyommaamericanum). Interestingly, there appears to be anincreased survival rate in domestic cats in thegeographic area of the Ozark Plateau. In this study,convenience blood samples from apparently healthyferal cats were microscopically evaluated for thepresence of C. felis merozoites. Positive samples weresubmitted for PCR confirmation by a commerciallaboratory. Results indicated a prevalence of 13% (4/32)in this population. Understanding the prevalence of C.felis infection in feral cats is central to evaluating theirpotential role as a reservoir for the disease and may alsofurther our understanding about the variablepathogenicity of this organism.

Introduction

Cytauxzoonosis is a hemoparasitic infection offelids that is characterized by a rapid course of diseasewith a high morbidity and mortality in the domestic cat(Felis catus). First reported in North America inMissouri (Wagner 1976) this emerging disease has sincebeen identified in wild and domestic felids from theMidwest to the mid-Atlantic states in the U.S.(Birkenheuer et al. 2006a; Haber et al. 2007; Shock etal. 2011; Tarigo et al. 2013; Zieman et al. 2017).Cytauxzoonosis is caused by an apicomplexanhematozoan parasite, Cytauxzoon felis (C. felis), that istransmitted by ixodid (hard shell) ticks. Although C.felis is thought to infect felids only, a closely relatedgenus Babesia causes tick-borne disease in a wide

variety of vertebrate hosts, including man. Thus C. felisbelongs to a family of hemoparasites that hasagricultural and zoonotic importance worldwide(Alvarado-Rybak et al. 2016; Wang et al. 2017). In theUnited States the principal vector of C. felis appears tobe the lone star tick (Amblyomma americanum)(Reichard et al. 2009). The American dog tick(Dermacentor variabilis) is also known to carry theprotozoan, but its ability to transmit the disease may belimited (Reichard et al. 2009). Both vectors haveextensive ranges that overlap with the currentdistribution of C. felis. Both tick vectors are found inArkansas (McAllister et al. 2016).

In North America, the bobcat (Lynx rufus) is theprimary sylvatic reservoir for this parasite. The reportedprevalence of infection in bobcats ranges from 7-70%(Birkenheur et al. 2008; Shock et al. 2011; Zieman et al.2017) and the natural infection in these animals isapparently self-limiting and asymptomatic. In thedomestic cat, on the other hand, the course of disease israpid and highly fatal. Clinical signs of cytauxzoonosisare non-specific and typically include acute onset offever, inappetance, lethargy or weakness, palor, icterus,and/or respiratory distress. Death occurs as a result ofmultiple organ failure caused by widespread vascularocclusion. With treatment, a 60% survival has beenreported (Cohn et al. 2011). Thus the domestic cat hashistorically been considered to be an aberrant or dead-end host.

The life cycle of C. felis is complex involving sexualreproduction within the tick vector, as well as asexualreplication (schizogony, or merogany) in the felid host(Fig. 1). The red blood cell inclusions, calledmerozoites, are the basis of the cytological screeningtest for the disease. Domestic cats that manage tosurvive the acute schizogenous phase of the disease donot completely clear the organism from their system.Instead they become chronic carriers and a potentialreservoir for the parasite. In these animals, a low levelparasitemia may persist for years following naturalinfection (Brown et al. 2008).

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Figure 1: Life cycle of Cytauxzoon felis. The acute schizogenousphase is typically fatal in the domestic cat. Hosts that survive developa chronic erythroparasitemia with merozoite-infected red blood cellsthat are the basis of cytologic screening. CCAL image from: TarigoJL, et al. (2013).

Interestingly, the pathogenicity of C. felis indomestic cats appears to vary between enzootic regions.In the 1990s reports began to emerge of pet cats thatsurvived infection with C. felis (Walker et al. 1995;Meinkoth et al. 2000). These early reports of subclinicaldisease were from cats that originated in the OzarkPlateau of Arkansas, Missouri, and Oklahoma. A 2007study of asymptomatic free-roaming cats enrolled intrap-neuter-release (TNR) programs on the east coastand in Tennessee found a low prevalence of subclinicalinfection, 0.3% (n=961) (Haber et al. 2007). Morerecently, a study in apparently healthy domestic cats inand around the Ozark Plateau of Oklahoma, Arkansas,and Missouri identified an overall disease prevalence of6.9% (n=902). Within this region, the prevalence ofinfection ranged from 3.4% in Oklahoma to 12.9% insouthern Missouri and 15.5% in northwest Arkansas(Rizzi et al. 2015).

The difference in the prevalence of inapparentcarriers between the eastern region of the U.S. (0.3%)and the Ozark Plateau (3.4-15.5%) may suggest theexistence of a less pathogenic strain of C. felis in theOzark region. To test this hypothesis, molecular studieshave explored the genetic variability and revisited thetaxonomy of these piroplasms (Brown et al. 2010;Shock et al. 2012; Schreeg et al. 2016; Pollard et al.2017). Other factors may influence the pathogenicity ofC. felis in domestic cats such as differences in the innate

immunity of the host or the dose of the infectiousinoculum. (Tarigo et al. 2013). It is possible that adifference in the biology of the tick vector affects thepathogenicity of the parasite. It is also possible that theincreasing availability of more sensitive diagnostic testsare simply increasing the frequency of detection ofasymptomatic carriers. In a recent review article oncytauxzoonosis in domestic cats, it was suggested thatnon-fatal, clinically inapparent infections should nolonger be considered rare; especially in enzooticregions. (Wang et al. 2017). Still, the epidemiology ofcytauxzoonosis is complex and gaps in ourunderstanding remain.

The purpose of this study was to measure theprevalence of C. felis in apparently healthy feral cats inRussellville Arkansas. Russellville is located in theArkansas River Valley, adjacent to the Ozark Plateau.Venous blood samples were microscopically evaluatedfor C. felis merozoites and positive samples weresubmitted for PCR confirmation by a commercialveterinary diagnostic laboratory. Measuring theprevalence of this infection in feral animals mayimprove our understanding of the epidemiology of thisdisease and the role of feral cats as a disease reservoir.

Materials and Methods

From April through August 2013, convenienceblood samples were obtained from 33 feral cats thatwere live-trapped as part of a wildlife science graduatestudy of the feral population in Russellville, Arkansas(Norman 2014). Consistent with institutionalrequirements at the time, all trapping and handlingprocedures were performed in accordance with theguidelines of the American Society of Mammalogists(Sikes et al. 2011). Briefly, a general exam wasperformed by the graduate student while the animal wasunder sedation with dexmeditomidine HCl(Dexdomitor®, Zoetis U.S.A.) The animal’s sex,approximate age based on dentition, weight,temperature and body condition score (BCS) wererecorded. Additional health information collectedincluded a visual inspection for ectoparasites, externalsigns of illness such as naso-ocular discharge, orevidence of diarrhea, and evidence of pregnancy orlactation. Approximately 0.5ml of venous blood wascollected in lithium heparin, orethylenediaminetetraacetic acid (EDTA) micro-vacutainers (Becton Dickenson Co., Franklin Lakes,N.J.). The sedation was reversed with atipamezole HCl(Antisedan®, Zoetis U.S.A) and the animal wasreleased following full recovery. The blood samples

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were chilled and transported in a cold pack back to thelab where they were used for whole blood analysis forfeline leukemia (FeLV) and feline immunodeficiencyvirus (FIV) as part of the primary research project.

Within 24 hours blood smears were made from theresidual blood for cytological screening for C. felis. Theslides were prepared in triplicate for each sample and airdried prior to staining. Two of the slides were stainedwith a Romanowsky stain (DipQuik, Jorgenson labs)and the third unstained blood smear was stored forfuture reference. Stained specimens were examinedunder oil immersion (1000x) for the presence of C. felismerozoites in erythrocytes. Fifty to 75 high power fields(hpf) were examined, focusing on the monolayer regionof the smear. The feather edge was also screened forschizonts. Although C. felis merozoites may present inseveral forms, only the signet and ‘safety pin’ formswere counted. The sample was considered positive if 5or more affected red blood cells were identified per 50hpf. This represents an estimated parasitemia of ≥ 0.05%; assuming an average of 200 rbc/hpf in themonolayer region of the blood smear.

Samples from the animals that were positive oncytology were submitted for confirmation testing by acommercial veterinary diagnostic laboratory (Idexxlaboratories, Westbrook, ME) where unstained bloodsmears were used for DNA extraction and polymerasechain reaction (PCR) analysis (C. felis RealPCR™,Idexx labs). One cytologically negative specimen wasalso submitted for PCR testing as a negative control.

The overall disease prevalence was calculated as theproportion of the specimens that were positive on bothcytology and PCR. Binomial confidence intervals werecalculated at the 95% confidence level.

The stated purpose of this study was to identify C.felis infections in apparently healthy feral cats. Due tothe non-specific signs associated with this disease andthe difficulty in assessing these signs in feral animals,the presence of fever (>102.5°F) was used as anexclusion criterion for identifying clinically affectedanimals.

Results

Stained blood smears from 33 feral cats wereexamined microscopically for the presence of C. felismerozoites. Six of the specimens tested positive oncytology. Unstained slides were available for 5 of thesesamples and they were subsequently submitted forconfirmation testing by PCR at a commercial veterinarydiagnostic laboratory. Four of the cytologically positivespecimens also tested positive on PCR (Table 1). This

indicates an overall prevalence of 13% (CL 95%, CI 3-32)

Three of the animals that tested positive for C. feliswere afebrile at the time of sample collection. The initialtemperature for one animal was not recorded; however,the general exam for that animal was normal. Thus all 4animals were considered to be inapparent carriers of C.felis. All of the infected animals were adults (> 6months). Three were males and one was a female.

As mentioned earlier, one cytologically negativespecimen was also submitted for PCR confirmation as anegative control (not shown in Table 1). This sampletested negative on PCR as well.

Discussion

This study measured the prevalence of C. felis inapparently healthy feral cats living in an exurbanenvironment in the Arkansas River Valley. The studyarea is located adjacent to the Ozark Plateau wherecytauxzoonosis is enzootic and the sylvatic reservoir(bobcat) and principal vector (lone star tick) arecommon. The prevalence of subclinical C. felis infectionin this population of feral cats was 13% (4/32; CL 95%,3 - 32). These findings are similar to levels previouslyreported in free roaming and pet cats from the OzarkHighlands of eastern OK (16.9%, 9.7-27.2), northwestAR (15.3%, 10.3-21.7), and southern Missouri (12.9%,6.1-24.0) (Rizzi et al. 2015).

The prevalence of cytauxzoonosis has been shownto vary within enzootic regions. For example, in centralOklahoma the reported prevalence (3.4%) was lowerthan that found in eastern Oklahoma (16.9%) (Rizzi etal. 2015; Nagamori et al. 2016). A recent study utilizedan ecological niche model to predict the distribution ofC. felis in domestic cats in Oklahoma, Missouri, and

Table 1. Results of cytology and PCR diagnostic testingfor Cytauxzoon felis in 33 feral cats. Overall prevalence(specimens positive on both tests) was 13% (4/32).Note: 5 of 6 samples that were positive on cytology wereavailable for PCR confirmation.

C. felis Cytology PCR* Cytology+ PCR

Positive 6 4 4

Negative 27 1 28

Total 33 5 32

Prevalence(95% CL)

18%(7 - 35)

13%(3 - 32)

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Arkansas (Mueller et al. 2013). The model usedconfirmed cytauxzoonosis case records, vector andsylvatic reservoir distribution data, and otherenvironmental factors to generate maps that predict highrisk areas for C. felis. Notwithstanding the author’scaution that limited data was available from Arkansas,the model predicted a lower prevalence of disease in theRiver Valley in Arkansas versus the surroundinghighlands of the Ozarks to the north and the Ouachitamountains to the south. The results of the current studywould suggest that C. felis is more evenly distributed inwestern Arkansas.

Although health assessment in feral animals is achallenging prospect, a general exam was performed onthe sedated animals in this study. None of the animalsthat tested positive for C. felis exhibited fever or otherexternal signs of disease consistent with felinecytauxzoonosis at the time of blood draw. Sinceinapparent carriers can remain parasitemic for aprolonged period of time, they present an increased riskof infection for naïve free roaming cats and a reservoirof disease in this exurban setting.

In addition to the small sample size in the currentstudy, there are inherent limitations for each of thediagnostic tests used (cytology, PCR) that should beconsidered when interpreting these results. Cytologicalscreening for merozoites in asymptomatic animals ischallenging in part because of the low levels ofparasitemia that are commonly encountered. Theerythrocytic merozoites of C. felis are small (1-2 µm)and are found in several different forms (signet, ‘safetypin’, punctate). The later can be difficult to distinguishfrom Howell Jolly bodies, stain precipitate and evendrying artifacts (Fig. 2). Further, some of the convenience

Figure 2. Cytauxzoon felis merozoite (black arrow) on a peripheralblood smear. The blue arrow indicates a Howell Jolly body. Blood isstained with a Romanowski differential stain (DipQuik). Oil(1000x).

blood samples used in this study were originallycollected in heparinized tubes. Heparin is not an idealanticoagulant for examining the morphology oferythrocytes as it can cause cellular distortion andvariable stain uptake. Thus cytological screening,although rapid and inexpensive, has limitations. PCRanalysis for parasitic DNA is a more sensitive andspecific test when compared to cytology. (Birkenheueret al. 2006b). Ideally, all samples (positive and negativeon cytology) would be confirmed by PCR. These testsare commercially available but expensive to run and aswith any test, false positives and false negatives arepossible. In this study, one sample was identified aspositive on cytology but tested negative on PCR.Because PCR is a more specific test than cytology, thesample was recorded as negative for C. felis (Table 1).

Conclusion

The current study measured a 13% prevalence ofCytauxzoon felis infections in apparently healthy feralcats living in Russellville, Arkansas. A comparable levelof infection was reported previously in asymptomaticcats in the adjacent Ozark Highlands region (Rizzi et al2015). Thus, the results of the current study suggest thatthe distribution of C. felis extends into the ArkansasRiver Valley at a similar level. The relatively highprevalence of C. felis reported here also supports thehypothesis that feral cats serve as a reservoir of infectionfor free roaming, naïve cats in exurban environmentswhere the sylvatic reservoir (bobcat) is less common.Further study of the epidemiology of cytauxzoonosis isneeded to assess whether the prevalence ofasymptomatic carriers reflects a change in host-parasiteinteraction.

Acknowledgments

The author is grateful to Ms. Catherine Normandand Dr. Rachael E. Urbanek for providing theconvenience blood samples used in this study, to Idexxlabs for providing PCR confirmation testing, and toArkansas Tech University for the laboratory support andfunds to defray the cost of publication. The authorwould also like to thank the anonymous JAAS reviewersfor their constructive criticism and suggestions forimproving this manuscript.

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Literature Cited

Alvarado-Rybak M, L Solano-Gallego, and J Millán.2016. A review of piroplasmid infections in wildcarnivores worldwide: importance for domesticanimal health and wildlife conservation. Parasites &Vectors 9:538.

Birkenheuer AJ, JA Le, AM Valenzisi, MD Tucker,MG Levy, and EB Breitschwerdt. 2006a.Cytauxzoon felis infection in cats in the Mid-Atlantic States: 34 Cases (1998-2004). Journal ofthe American Veterinary Medical Association228:568-571.

Birkenheuer AJ, H Marr, AR Alleman, MG Levy,and EB Breitschwerdt. 2006b. Development andevaluation of a PCR assay for the detection ofCytauxzoon felis DNA in feline blood samples.Veterinary Parasitology 137:144-149.

Birkenheuer AJ, HS Marr, C Warren, AE Acton,EM Mucker, JG Humphreys, and MD Tucker.2008. Cytauxzoon felis infections are present inbobcats (Lynx rufus) in a region wherecytauxzoonosis is not recognized in domestic cats.Veterinary Parasitology 153:126-130.

Brown HM, KS Latimer, LE Erikson, ME Cashwell,JO Britt, and DS Peterson. 2008. Detection ofpersistent Cytauxzoon felis infection by polymerasechain reaction in three asymptomatic domestic cats.Journal of Veterinary Diagnostic Investigation20:485-488.

Brown HM, JM Lockhart, KS Latimer, and DSPeterson. 2010. Identification and geneticcharacterization of Cytauxzoon felis inasymptomatic domestic cats and bobcats.Veterinary Parasitology 172:311-316.

Cohn LA, AJ Birkenheuer, JD Brunker, ER Ratcliff,and AW Craig. 2011. Efficacy of atovaquone andazithromycin or imidocarb dipropionate in cats withacute cytauxzoonosis. Journal of VeterinaryInternal Medicine 25:55-60.

Haber MD, MD Tucker, HS Marr, JK Levy, JBurgess, MR Lappin, and AJ Birkenheuer. 2007.The detection of Cytauxzoon felis in apparentlyhealthy free-roaming cats in the U.S.A. Journal ofVeterinary Parasitology Vol. 146:316-320.

McAllister CT, LA Durden, and HW Robison. 2016.The ticks (Arachnida: Acari: Ixodida) of Arkansas.Journal of the Arkansas Academy of Sciences70:141-154.

Meinkoth J, AA Kocan, L Whitworth, G Murphy,JC Fox, and JP Woods. 2000. Cats survivingnatural infection with Cytauxzoon felis: 18 cases(1997–1998). Journal of Veterinary InternalMedicine 14:521-525.

Mueller EK, KA Baum, M Papeş, LA Cohn, AK Cowell, and MV Reichard. 2013. Potentialecological distribution of Cytauxzoon felis indomestic cats in Oklahoma, Missouri, andArkansas. Veterinary Parasitology 192:104-110.

Nagamori Y, JE Slovak, and MV Reichard. 2016.Prevalence of Cytauxzoon felis infection in healthyfree-roaming cats in north-central Oklahoma andcentral Iowa. Journal of Feline Medicine andSurgery Open Reports 2:1-4.

Norman C. 2014. Feral cat population dynamics, multi-scale habitat selection, and disease prevalence in anexurban landscape. [MS thesis]. Russellville (AR):Arkansas Tech University. 114 p.

Pollard DA, MV Reichard, LA Cohn, AM James, andPJ Holman. 2017. Genetic variability of clonedCytauxzoon felis ribosomal RNA ITS1 and ITS2genomic regions from domestic cats with variedclinical outcomes from five states. VeterinaryParasitology 244:136-143.

Reichard MV, JH Meinkoth, AC Edwards, TASnider, KM Kocan, EF Blouin, and SE Little.2009. Transmission of Cytauxzoon felis to adomestic cat by Amblyomma americanum.Veterinary Parasitology 161:110-115.

Rizzi TE, MV Reichard, LA Cohn, AJ Birkenheuer,JD Taylor, and JH Meinkoth. 2015. Prevalence ofCytauxzoon felis infection in healthy cats fromenzootic areas in Arkansas, Missouri, andOklahoma. Parasites & Vectors 8:13.

Schreeg ME, HS Marr, JL Tarigo, LA Cohn, DMBird, EH Scholl, MG Levy, et al. 2016.Mitochondrial genome sequences and structures aidin the resolution of Piroplasmida phylogeny. PLoSONE 11: e0165702.

Shock BC, SM Murphy, LL Patton, PM Shock, COlfenbuttel, J Beringer, S Prange, et al. 2011.Distribution and prevalence of Cytauxzoon felis inbobcats (Lynx rufus), the natural reservoir, and otherwild felids in thirteen states. VeterinaryParasitology 175:325-330.

127

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Published by Arkansas Academy of Science, 2018

C.H. Jacobs

Journal of the Arkansas Academy of Science, Vol. 72, 2018128

Shock BC, AJ Birkenheuer, LL Patton, COlfenbuttel, J Beringer, DM Grove, M Peek, etal. 2012. Variation in the ITS-1 and ITS-2 rRNAgenomic regions of Cytauxzoon felis from bobcatsand pumas in the eastern United States andcomparison with sequences from domestic cats.Veterinary Parasitology 190:29-35.

Sikes RS, WL Gannon, and the Animal Care and UseCommittee 1 of the American Society ofMammalogists. 2011. Guidelines of the AmericanSociety of Mammalogists for the use of wildmammals in research. Journal of Mammalogy92:235-253.

Tarigo JL, EH Scholl, DM Bird, CC Brown, LACohn, GA Dean, MG Levy, et al. 2013. A Novelcandidate vaccine for cytauxzoonosis inferred fromcomparative Apicomplexan genomics. PloS One 8:e71233.

Wagner JE. 1976. A fatal cytauxzoonosis-like diseasein cats. Journal of the American Veterinary MedicalAssociation. 168:585-588.

Walker DB and RL Cowell. 1995. Survival of adomestic cat with naturally acquiredcytauxzoonosis. Journal of the American VeterinaryMedical Association 206:1363-1365.

Wang JL, TT Li, GH Liu, XQ Zhu, and C Yao. 2017.Two tales of Cytauxzoon felis infections in domesticcats. Clinical Microbiology Reviews 30:861-884.

Zieman EA, FA Jiménez, and CK Nielsen. 2017.Concurrent examination of bobcats and ticks revealshigh prevalence of Cytauxzoon felis in SouthernIllinois. Journal of Parasitology 103:343-348.

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